A new β-rhamnoslytransferase Cps23FT from Streptococcus pneumonia serotype 23F was expressed and characterized. Its enzymatic activity and function were confirmed for the first time by utilizing enzymatically prepared dTDP-Rha and chemically synthesized Glcα-PP-(CH2)11-OPh as substrates. This reaction gave the desired disaccharide Rhaβ-1,4-Glcα-PP-(CH2)11-OPh in a good isolated yield (67%), suggesting the potential of Cps23FT as a tool enzyme for the synthesis of complex oligosaccharides containing difficult β-rhamnosyl linkages. Furthermore, site-directed mutagenesis of Cps23FT disclosed that its 271DKD273 motif was critical for the enzymatic activity and most likely the binding site for the required divalent metal cation.